Kristen Frenzel

Transmembrane Neuregulins Interact with LIM Kinase 1, a Cytoplasmic Protein Kinase Implicated in Development of Visuospatial Cognition

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-α did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

Male fertility is dependent on dipeptidase activity of testis ACE.

c Figure 2 ACE overexpression in CHO and HEK cells does not affect the shedding of multiple GPI-anchored proteins. (a) CHO cells were stably transfected with vector alone (mock), full-length wild-type (FL-ACE) or GPI-ACE10. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Results are means ± s.d. (n = 3) and are expressed as a percentage of activity shed into media of mock-transfected cells. (b) HEK cells stably transfected with either doppel or prion protein were transiently transfected with vector alone (mock), FL-ACE or GPI-ACE. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Doppel and prion protein shed into media were determined by immunoblotting followed by densitometric analysis. Results are means ± s.d. (n = 3 or 6 (alkaline phosphatase)) and are expressed as a percentage of protein shed into the media of mock-transfected cells. (c) ACE activity determined using BzGly-His-Leu as substrate in lysates from the HEK cells transiently transfected with vector alone (mock), FL-ACE or GPI-ACE as in b. Results are means ± s.d. (n = 3).

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts.

Neuregulin‐1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin‐1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N‐terminal fragment containing the bioactive EGF‐like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140‐kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X‐100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low‐density fractions enriched in PSD‐95, a known lipid raft protein. In transfected cells expressing the neuregulin I‐β1a or the III‐β1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X‐100, and neuregulin proproteins and C‐terminal fragments were detected in lipid raft fractions. In contrast, the III‐β1a N‐terminal fragment was detected only in the detergent‐soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.

Six Truisms Concerning ACE and the Renin-Angiotensin System Educed From the Genetic Analysis of Mice

This Review is part of a thematic series on Angiotensin Converting Enzyme , which includes the following articles: Six Truisms Concerning ACE and the Renin-Angiotensin System Educed From the Genetic Analysis of Mice ACE and Vascular Remodeling ACE II in the Heart and the Kidney ACE Signaling ACE Polymorphisms Rudi Bussi Editors The history of the renin-angiotensin system (RAS) is one of marvelous discoveries extending from Robert Tigerstedt’s naming renin in 1898 to the present time; biochemists, physiologists, pharmacologists, and practicing clinicians have all combined to describe the physiologic implications of converting angiotensinogen into angiotensin II. Indeed, one may argue that the clinical development of ACE inhibitors and angiotensin II receptor antagonists has benefited humankind to a level seen only with the development of antibiotics and steroids. As we begin the twenty-first century, it is worthwhile to summarize the state of our knowledge concerning ACE and angiotensin II. In doing, we benefit from a whole class of experiments not available to those writing reviews even 10 years ago: the revolution in our ability to genetically manipulate the mouse as an experimental model. This is due to the widespread application of gene targeting by homologous recombination in embryonic stem cells. As is widely appreciated, this technology can produce a knockout mouse lacking any particular gene. Less appreciated are the full capabilities of this methodology which can be summarized as: if it can be dreamed, it can be done. Gene targeting can be used to create point mutations, duplicate a gene, and modify the expression pattern of a protein almost as easily as creating knockout mice null for a particular protein. We, and others, have used gene targeting in mice to create modifications in the renin-angiotensin system of a sort not seen in humans. While any single experiment may be assailed as …

Establishing the Role of Angiotensin-Converting Enzyme in Renal Function and Blood Pressure Control through the Analysis of Genetically Modified Mice

Angiotensin II is a vasoconstrictor and a hypertensive peptide that binds to the AT1 receptor and, through both direct and indirect mechanisms, induces salt reabsorption. Also, angiotensin II is thought to be a profibrotic and proproliferative peptide; abundant evidence now suggests that angiotensin

Newer Approaches to Genetic Modeling in Mice : Tissue-Specific Protein Expression as Studied Using Angiotensin-Converting Enzyme (ACE)

Virtually all scientists are aware of the tremendous progress in genetic modeling brought about by targeted homologous recombination in embryonic stem cells. Often called a “knockout mouse”, the ability to modify the mouse genome has created animal models for a large number of disease processes. Typically, targeted genetic modifications are used to inactivate a gene resulting in a mouse null for the corresponding protein. While this approach has been tremendously useful, the most recent work in this area makes use of more subtle genetic modifications to probe the functional role of a protein in an organ-specific or developmental fashion. This review will touch on work from my group and several other laboratories that are using newer approaches to gene targeting with the goal of creating mouse models that ask questions not addressable through the simple inactivation of a gene.